Novel human kinases and polynucleotides encoding the same

ABSTRACT

Novel human polynucleotide and polypeptide sequences are disclosed that can be used in therapeutic, diagnostic, and pharmacogenomic applications.

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/252,011, which was filed on Nov. 20, 2000 and isherein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] The present invention relates to the discovery, identification,and characterization of novel human polynucleotides encoding proteinssharing sequence similarity with animal kinases. The inventionencompasses the described polynucleotides, host cell expression systems,the encoded proteins, fusion proteins, polypeptides and peptides,antibodies to the encoded proteins and peptides, and geneticallyengineered animals that either lack or overexpress the disclosed genes,antagonists and agonists of the proteins, and other compounds thatmodulate the expression or activity of the proteins encoded by thedisclosed genes, which can be used for diagnosis, drug screening,clinical trial monitoring, the treatment of diseases and disorders, andcosmetic or nutriceutical applications.

2. BACKGROUND OF THE INVENTION

[0003] Kinases mediate the phosphorylation of a wide variety of proteinsand compounds in the cell. In conjunction with phosphatases, kinases areinvolved in a range of regulatory pathways. Given the physiologicalimportance of kinases, they have been subject to intense scrutiny andare proven drug targets.

3. SUMMARY OF THE INVENTION

[0004] The present invention relates to the discovery, identification,and characterization of nucleotides that encode novel human proteins andthe corresponding amino acid sequences of these proteins.. The novelhuman proteins (NHPs) described for the first time herein sharestructural similarity with animal kinases, including, but not limitedto, receptor tyrosine kinases (SEQ ID NOS:1-2 show particular similarityto NEK family kinases, and SEQ ID NOS:3-5 are particularly similar tocalcium and calmodulin dependent kinases as well as sequences encodingPK 80), and serine-threonine kinases. The described NHPs encode novelkinases having homologues and orthologs across a range of phyla andspecies.

[0005] The novel human polynucleotides described herein encode openreading frames (ORFs) encoding proteins of 692 and 817 amino acids inlength (see respectively SEQ ID NOS:2 and 4).

[0006] The invention also encompasses agonists and antagonists of thedescribed NHPs, including small molecules, large molecules, mutant NHPs,or portions thereof, that compete with native NHP, peptides, andantibodies, as well as nucleotide sequences that can be used to inhibitthe expression of the described NHPs (e.g., antisense and ribozymemolecules, and open reading frame or regulatory sequence replacementconstructs) or to enhance the expression of the described NHPs (e.g.,expression constructs that place the described polynucleotide under thecontrol of a strong promoter system), and transgenic animals thatexpress a NHP sequence, or “knock-outs” (which can be conditional) thatdo not express a functional NHP. Knock-out mice can be produced inseveral ways, one of which involves the use of mouse embryonic stemcells (“ES cells”) lines that contain gene trap mutations in a murinehomolog of at least one of the described NHPs. When the unique NHPsequences described in SEQ ID NOS:1-5 are “knocked-out” they provide amethod of identifying phenotypic expression of the particular gene aswell as a method of assigning function to previously unknown genes. Inaddition, animals in which the unique NHP sequences described in SEQ IDNOS:1-5 are “knocked-out” provide a unique source in which to elicitantibodies to homologous and orthologous proteins that would have beenpreviously viewed by the immune system as “self” and therefore wouldhave failed to elicit significant antibody responses. To these ends,gene trapped knockout ES cells have been generated in murine homologs ofthe described NHPs.

[0007] Additionally, the unique NHP sequences described in SEQ IDNOS:1-5 are useful for the identification of protein coding sequence andmapping a unique gene to a particular chromosome (the gene encoding SEQID NOS:1-2 is apparently encoded on human chromosome 17, see GENBANKaccession no. AC010761, and the gene encoding SEQ ID NOS:3-5 isapparently encoded on human chromosome 3, see GENBANK accession no.AC068979). These sequences identify biologically verified exon splicejunctions as opposed to splice junctions that may have beenbioinformatically predicted from genomic sequence alone. The sequencesof the present invention are also useful as additional DNA markers forrestriction fragment length polymorphism (RFLP) analysis, and inforensic biology.

[0008] Further, the present invention also relates to processes foridentifying compounds that modulate, i.e., act as agonists orantagonists, of NHP expression and/or NHP activity that utilize purifiedpreparations of the described NHPs and/or NHP products, or cellsexpressing the same. Such compounds can be used as therapeutic agentsfor the treatment of any of a wide variety of symptoms associated withbiological disorders or imbalances.

4. DESCRIPTION OF THE SEQUENCE LISTING AND FIGURES

[0009] The Sequence Listing provides the sequence of the novel humanORFs encoding the described novel human kinase proteins. SEQ ID NO:5describes a NHP ORF and flanking sequences.

5. DETAILED DESCRIPTION OF THE INVENTION

[0010] The NHPs described for the first time herein are novel proteinsthat are expressed in, inter alia, human cell lines and pituitary,thymus, spleen, lymph node, bone marrow, trachea, kidney, prostate,testis, thyroid, adrenal gland, pancreas, salivary gland, stomach, smallintestine, skeletal muscle, heart, uterus, placenta, adipose, skin,bladder, rectum, pericardium, ovary, fetal kidney, fetal lung, gallbladder, tongue, aorta, 6-, 9-, and 12-week embryos, adenocarcinoma,osteosarcoma, and embryonic carcinoma cells (SEQ ID NOS:1-2). SEQ IDNOS:3-5 were predominantly expressed in fetal brain, brain, spinal cord,thymus, lymph node, trachea, lung, prostate, testis, thyroid, adrenalgland, stomach, small intestine, skeletal muscle, uterus, placenta,mammary gland, skin, bladder, pericardium, hypothalamus, fetal kidney,fetal lung, tongue, aorta, 6-, 9-, and 12-week embryos, and embryoniccarcinoma cells.

[0011] The described sequences were compiled from sequences available inGENBANK, and cDNAs generated from pituitary, lymph node, mammary gland,brain, adrenal gland, fetus, and testis mRNAs (Edge Biosystems,Gaithersburg, Md.).

[0012] The present invention encompasses the nucleotides presented inthe Sequence Listing, host cells expressing such nucleotides, theexpression products of such nucleotides, and: (a) nucleotides thatencode mammalian homologs of the described genes, including thespecifically described NHPs, and the NHP products; (b) nucleotides thatencode one or more portions of an NHP that correspond to functionaldomains, and the polypeptide products specified by such nucleotidesequences, including, but not limited to, the novel regions of anyactive domain(s); (c) isolated nucleotides that encode mutant versions,engineered or naturally occurring, of the described NHPs in which all ora part of at least one domain is deleted or altered, and the polypeptideproducts specified by such nucleotide sequences, including, but notlimited to, soluble proteins and peptides in which all or a portion ofthe signal sequence is deleted; (d) nucleotides that encode chimericfusion proteins containing all or a portion of a coding region of a NHP,or one of its domains (e.g., a receptor/ligand binding domain, accessoryprotein/self-association domain, etc.) fused to another peptide orpolypeptide; or (e) therapeutic or diagnostic derivatives of thedescribed polynucleotides such as oligonucleotides, antisensepolynucleotides, ribozymes, dsRNA, or gene therapy constructs comprisinga sequence first disclosed in the Sequence Listing.

[0013] As discussed above, the present invention includes the human DNAsequences presented in the Sequence Listing (and vectors comprising thesame), and additionally contemplates any nucleotide sequence encoding acontiguous NHP open reading frame (ORF) that hybridizes to a complementof a DNA sequence presented in the Sequence Listing under highlystringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1× SSC/0.1% SDS at 68° C. (Ausubel et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3)and encodes a functionally equivalent expression product. Additionally,contemplated are any nucleotide sequences that hybridize to thecomplement of a DNA sequence that encodes and expresses an amino acidsequence presented in the Sequence Listing under moderately stringentconditions, e.g., washing in 0.2× SSC/0.1% SDS at 42° C. (Ausubel etal., 1989, supra), yet still encode a functionally equivalent NHPproduct. Functional equivalents of a NHP include naturally occurringNHPs present in other species and mutant NHPs whether naturallyoccurring or engineered (by site directed mutagenesis, gene shuffling,and/or directed evolution, as described in, for example, U.S. Pat. Nos.5,837,458 or 5,723,323, both of which are herein incorporated byreference). The invention also includes degenerate nucleic acid variantsof the disclosed NHP polynucleotide sequences.

[0014] Additionally contemplated are polynucleotides encoding NHP ORFs,or their functional equivalents, encoded by polynucleotide sequencesthat are about 99, 95, 90, or about 85 percent similar to correspondingregions of a NHP (as measured by BLAST sequence comparison analysisusing, for example, the GCG sequence analysis package, as describedherein, using default parameters).

[0015] The invention also includes nucleic acid molecules, preferablyDNA molecules, that hybridize to, and are therefore the complements of,the described NHP encoding polynucleotides. Such hybridizationconditions can be highly stringent or less highly stringent, asdescribed herein. In instances where the nucleic acid molecules aredeoxyoligonucleotides (“DNA oligos”), such molecules are generally about16 to about 100 bases long, or about 20 to about 80 bases long, or about34 to about 45 bases long, or any variation or combination of sizesrepresented therein that incorporate a contiguous region of sequencefirst disclosed in the Sequence Listing. Such oligonucleotides can beused in conjunction with the polymerase chain reaction (PCR) to screenlibraries, isolate clones, and prepare cloning and sequencing templates,etc.

[0016] Alternatively, such NHP oligonucleotides can be used ashybridization probes for screening libraries, and assessing geneexpression patterns (particularly using a microarray or high-throughput“chip” format). Additionally, a series of the described NHPoligonucleotide sequences, or the complements thereof, can be used torepresent all or a portion of the described NHP sequences. Anoligonucleotide or polynucleotide sequence first disclosed in at least aportion of one or more of the sequences of SEQ ID NOS:1-5 can be used asa hybridization probe in conjunction with a solid supportmatrix/substrate (resins, beads, membranes, plastics, polymers, metal ormetallized substrates, crystalline or polycrystalline substrates, etc.).Of particular note are spatially addressable arrays (i.e., gene chips,microtiter plates, etc.) of oligonucleotides and polynucleotides, orcorresponding oligopeptides and polypeptides, wherein at least one ofthe biopolymers present on the spatially addressable array comprises anoligonucleotide or polynucleotide sequence first disclosed in at leastone of the sequences of SEQ ID NOS:1-5, or an amino acid sequenceencoded thereby. Methods for attaching biopolymers to, or synthesizingbiopolymers on, solid support matrices, and conducting binding studiesthereon are disclosed in, inter alia, U.S. Pat. Nos. 5,700,637,5,556,752, 5,744,305, 4,631,211, 5,445,934, 5,252,743, 4,713,326,5,424,186, and 4,689,405, the disclosures of which are hereinincorporated by reference in their entirety.

[0017] Addressable arrays comprising sequences first disclosed in SEQ IDNOS:1-5 can be used to identify and characterize the temporal and tissuespecific expression of a gene. These addressable arrays incorporateoligonucleotide sequences of sufficient length to confer the requiredspecificity, yet be within the limitations of the production technology.The length of these probes is within a range of between about 8 to about2000 nucleotides. Preferably the probes consist of 60 nucleotides andmore preferably 25 nucleotides from the sequences first disclosed in SEQID NOS:1-5.

[0018] For example, a series of the described oligonucleotide sequences,or the complements thereof, can be used in chip format to represent allor a portion of the described sequences. The oligonucleotides, typicallybetween about 16 to about 40 (or any whole number within the statedrange) nucleotides in length can partially overlap each other and/or thesequence may be represented using oligonucleotides that do not overlap.Accordingly, the described polynucleotide sequences shall typicallycomprise at least about two or three distinct oligonucleotide sequencesof at least about 8 nucleotides in length that are each first disclosedin the described Sequence Listing. Such oligonucleotide sequences canbegin at any nucleotide present within a sequence in the SequenceListing and proceed in either a sense (5′-to-3′) orientation vis-a-visthe described sequence or in an antisense orientation.

[0019] Microarray-based analysis allows the discovery of broad patternsof genetic activity, providing new understanding of gene functions andgenerating novel and unexpected insight into transcriptional processesand biological mechanisms. The use of addressable arrays comprisingsequences first disclosed in SEQ ID NOS:1-5 provides detailedinformation about transcriptional changes involved in a specificpathway, potentially leading to the identification of novel componentsor gene functions that manifest themselves as novel phenotypes.

[0020] Probes consisting of sequences first disclosed in SEQ ID NOS:1-5can also be used in the identification, selection and validation ofnovel molecular targets for drug discovery. The use of these uniquesequences permits the direct confirmation of drug targets andrecognition of drug dependent changes in gene expression that aremodulated through pathways distinct from the intended target of thedrug. These unique sequences therefore also have utility in defining andmonitoring both drug action and toxicity.

[0021] As an example of utility, the sequences first disclosed in SEQ IDNOS:1-5 can be utilized in microarrays or other assay formats, to screencollections of genetic material from patients who have a particularmedical condition. These investigations can also be carried out usingthe sequences first disclosed in SEQ ID NOS:1-5 in silico and bycomparing previously collected genetic databases and the disclosedsequences using computer software known to those in the art.

[0022] Thus the sequences first disclosed in SEQ ID NOS:1-5 can be usedto identify mutations associated with a particular disease and also indiagnostic and/or prognostic assays.

[0023] Although the presently described sequences have been specificallydescribed using nucleotide sequence, it should be appreciated that eachof the sequences can uniquely be described using any of a wide varietyof additional structural attributes, or combinations thereof. Forexample, a given sequence can be described by the net composition of thenucleotides present within a given region of the sequence in conjunctionwith the presence of one or more specific oligonucleotide sequence(s)first disclosed in the SEQ ID NOS:1-5. Alternatively, a restriction mapspecifying the relative positions of restriction endonuclease digestionsites, or various palindromic or other specific oligonucleotidesequences can be used to structurally describe a given sequence. Suchrestriction maps, which are typically generated by widely availablecomputer programs (e.g., the University of Wisconsin GCG sequenceanalysis package, SEQUENCHER 3.0, Gene Codes Corp., Ann Arbor, Mich.,etc.), can optionally be used in conjunction with one or more discretenucleotide sequence(s) present in the sequence that can be described bythe relative position of the sequence relative to one or more additionalsequence(s) or one or more restriction sites present in the disclosedsequence.

[0024] For oligonucleotide probes, highly stringent conditions mayrefer, e.g., to washing in 6× SSC/0.05% sodium pyrophosphate at 37° C.(for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), or 60° C. (for 23-base oligos). These nucleic acid moleculesmay encode or act as NHP gene antisense molecules, useful, for example,in NHP gene regulation (and/or as antisense primers in amplificationreactions of NHP gene nucleic acid sequences). With respect to NHP generegulation, such techniques can be used to regulate biologicalfunctions. Further, such sequences can be used as part of ribozymeand/or triple helix sequences that are also useful for NHP generegulation.

[0025] Inhibitory antisense or double stranded oligonucleotides canadditionally comprise at least one modified base moiety that is selectedfrom the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

[0026] The antisense oligonucleotide can also comprise at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

[0027] In yet another embodiment, the antisense oligonucleotide willcomprise at least one modified phosphate backbone selected from thegroup including, but not limited to, a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and aformacetal or analog thereof.

[0028] In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330). Alternatively, double stranded RNA can be used todisrupt the expression and function of a targeted NHP.

[0029] Oligonucleotides of the invention can be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesized(Stein et al., 1988, Nucl. Acids Res. 16:3209), and methylphosphonateoligonucleotides can be prepared by use of controlled pore glass polymersupports (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451),etc.

[0030] Low stringency conditions are well-known to those of skill in theart, and will vary predictably depending on the specific organisms fromwhich the library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual (and periodic updates thereof),Cold Spring Harbor Press, N.Y.; and Ausubel et al., 1989, supra.

[0031] Alternatively, suitably labeled NHP nucleotide probes can be usedto screen a human genomic library using appropriately stringentconditions or by PCR. The identification and characterization of humangenomic clones is helpful for identifying polymorphisms (including, butnot limited to, nucleotide repeats, microsatellite alleles, singlenucleotide polymorphisms, or coding single nucleotide polymorphisms),determining the genomic structure of a given locus/allele, and designingdiagnostic tests. For example, sequences derived from regions adjacentto the intron/exon boundaries of the human gene can be used to designprimers for use in amplification assays to detect mutations within theexons, introns, splice sites (e.g., splice acceptor and/or donor sites),etc., that can be used in diagnostics and pharmacogenomics.

[0032] For example, the present sequences can be used in restrictionfragment length polymorphism (RFLP) analysis to identify specificindividuals. In this technique, an individuals genomic DNA is digestedwith one or more restriction enzymes, and probed on a Southern blot toyield unique bands for identification (as generally described in U.S.Pat. No. 5,272,057, incorporated herein by reference). In addition, thesequences of the present invention can be used to provide polynucleotidereagents, e.g., PCR primers, targeted to specific loci in the humangenome, which can enhance the reliability of DNA-based forensicidentifications by, for example, providing another “identificationmarker” (i.e., another DNA sequence that is unique to a particularindividual). Actual base sequence information can be used foridentification as an accurate alternative to patterns formed byrestriction enzyme generated fragments.

[0033] Further, a NHP gene homolog can be isolated from nucleic acidfrom an organism of interest by performing PCR using two degenerate or“wobble” oligonucleotide primer pools designed on the basis of aminoacid sequences within the NHP products disclosed herein. The templatefor the reaction may be total RNA, mRNA, and/or cDNA obtained by reversetranscription of mRNA prepared from, for example, human or non-humancell lines or tissue known to express or suspected of expressing anallele of a NHP gene.

[0034] The PCR product can be subcloned and sequenced to ensure that theamplified sequences represent the sequence of the desired NHP gene. ThePCR fragment can then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment can be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment can be used to isolate genomicclones via the screening of a genomic library.

[0035] PCR technology can also be used to isolate full length cDNAsequences. For example, RNA can be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown to express, or suspected of expressing, a NHP gene). A reversetranscription (RT) reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis. The resultingRNA/DNA hybrid may then be “tailed” using a standard terminaltransferase reaction, the hybrid may be digested with RNase H, andsecond strand synthesis may then be primed with a complementary primer.Thus, cDNA sequences upstream of the amplified fragment can be isolated.For a review of cloning strategies that can be used, see e.g., Sambrooket al., 1989, supra.

[0036] A cDNA encoding a mutant NHP sequence can be isolated, forexample, by using PCR. In this case, the first cDNA strand may besynthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolatedfrom tissue known to express, or suspected of expressing, a mutant NHPallele, in an individual putatively carrying a mutant NHP allele, and byextending the new strand with reverse transcriptase. The second strandof the cDNA is then synthesized using an oligonucleotide that hybridizesspecifically to the 5′ end of the normal sequence. Using these twoprimers, the product is then amplified via PCR, optionally cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell-known to those of skill in the art. By comparing the DNA sequenceof the mutant NHP allele to that of a corresponding normal NHP allele,the mutation(s) responsible for the loss or alteration of function ofthe mutant NHP gene product can be ascertained.

[0037] Alternatively, a genomic library can be constructed using DNAobtained from an individual suspected of carrying, or known to carry, amutant NHP allele (e.g., a person manifesting a NHP-associated phenotypesuch as, for example, immune disorders, obesity, high blood pressure,etc.), or a cDNA library can be constructed using RNA from a tissueknown to express, or suspected of expressing, a mutant NHP allele. Anormal NHP sequence, or any suitable fragment thereof, can then belabeled and used as a probe to identify the corresponding mutant NHPallele in such libraries. Clones containing mutant NHP sequences canthen be purified and subjected to sequence analysis according to methodswell-known to those skilled in the art.

[0038] Additionally, an expression library can be constructed utilizingcDNA synthesized from, for example, RNA isolated from a tissue known toexpress, or suspected of expressing, a mutant NHP allele in anindividual suspected of carrying, or known to carry, such a mutantallele. In this manner, gene products made by the putatively mutanttissue may be expressed and screened using standard antibody screeningtechniques in conjunction with antibodies raised against a normal NHPproduct, as described below (for screening techniques, see, for example,Harlow and Lane, eds., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Press, Cold Spring Harbor).

[0039] Additionally, screening can be accomplished by screening withlabeled NHP fusion proteins, such as, for example, alkalinephosphatase-NHP or NHP-alkaline phosphatase fusion proteins. In caseswhere a NHP mutation results in an expression product with alteredfunction (e.g., as a result of a missense or a frameshift mutation),polyclonal antibodies to a NHP are likely to cross-react with acorresponding mutant NHP expression product. Library clones detected viatheir reaction with such labeled antibodies can be purified andsubjected to sequence analysis according to methods well-known in theart.

[0040] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721, 5,837,458, 6,117,679, and 5,723,323, which areherein incorporated by reference in their entirety.

[0041] The invention also encompasses: (a) DNA vectors that contain anyof the foregoing NHP coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingNHP coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences (for example,baculovirus as described in U.S. Pat. No. 5,869,336 herein incorporatedby reference); (c) genetically engineered host cells that contain any ofthe foregoing NHP coding sequences operatively associated with aregulatory element that directs the expression of the coding sequencesin the host cell; and (d) genetically engineered host cells that expressan endogenous NHP sequence under the control of an exogenouslyintroduced regulatory element (i.e., gene activation). As used herein,regulatory elements include, but are not limited to, inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include, but are not limited to, thecytomegalovirus,(hCMV) immediate early gene, regulatable, viral elements(particularly retroviral LTR promoters), the early or late promoters ofSV40 and adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage lambda, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase (PGK), the promoters of acid phosphatase, and the promoters ofthe yeast α-mating factors.

[0042] Where, as in the present instance, some of the described NHPpeptides or polypeptides are thought to be cytoplasmic or nuclearproteins (although processed forms or fragments can be secreted ormembrane associated), expression systems can be engineered that producesoluble derivatives of a NHP (corresponding to a NHP extracellularand/or intracellular domains, or truncated polypeptides lacking one ormore hydrophobic domains) and/or NHP fusion protein products (especiallyNHP-Ig fusion proteins, i.e., fusions of a NHP domain to an IgFc), NHPantibodies, and anti-idiotypic antibodies (including Fab fragments) thatcan be used in therapeutic applications. Preferably, the aboveexpression systems are engineered to allow the desired peptide orpolypeptide to be recovered from the culture media.

[0043] The present invention also encompasses antibodies andanti-idiotypic antibodies (including Fab fragments), antagonists andagonists of a NHP, as well as compounds or nucleotide constructs thatinhibit expression of a NHP sequence (transcription factor inhibitors,antisense and ribozyme molecules, or open reading frame sequence orregulatory sequence replacement constructs), or promote the expressionof a NHP (e.g., expression constructs in which NHP coding sequences areoperatively associated with expression control elements such aspromoters, promoter/enhancers, etc.).

[0044] The NHPs or NHP peptides, NHP fusion proteins, NHP nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant NHPs or inappropriately expressed NHPs for thediagnosis of disease. The NHP proteins or peptides, NHP fusion proteins,NHP nucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs (or high throughput screening ofcombinatorial libraries) effective in the treatment of the symptomaticor phenotypic manifestations of perturbing the normal function of a NHPin the body. The use of engineered host cells and/or animals can offeran advantage in that such systems allow not only for the identificationof compounds that bind to the endogenous receptor/ligand of a NHP, butcan also identify compounds that trigger NHP-mediated activities orpathways.

[0045] Finally, the NHP products can be used as therapeutics. Forexample, soluble derivatives such as NHP peptides/domains correspondingto NHPs, NHP fusion protein products (especially NHP-Ig fusion proteins,i.e., fusions of a NHP, or a domain of a NHP, to an IgFc), NHPantibodies and anti-idiotypic antibodies (including Fab fragments),antagonists or agonists (including compounds that modulate or act ondownstream targets in a NHP-mediated pathway) can be used to directlytreat diseases or disorders. For instance, the administration of aneffective amount of soluble NHP, or a NHP-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the NHP could activateor effectively antagonize the endogenous NHP or a protein interactivetherewith. Nucleotide constructs encoding such NHP products can be usedto genetically engineer host cells to express such products in vivo;these genetically engineered cells function as “bioreactors” in the bodydelivering a continuous supply of a NHP, a NHP peptide, or a NHP fusionprotein to the body. Nucleotide constructs encoding functional NHPs,mutant NHPs, as well as antisense and ribozyme molecules can also beused in “gene therapy” approaches for the modulation of NHP expression.Thus, the invention also encompasses pharmaceutical formulations andmethods for treating biological disorders.

[0046] Various aspects of the invention are described in greater detailin the subsections below.

5.1 THE NHP SEQUENCES

[0047] The cDNA sequences and corresponding deduced amino acid sequencesof the described NHPs are presented in the Sequence Listing.

[0048] Expression analysis has provided evidence that the described NHPscan be expressed in a range of human tissues, as described in greaterdetail herein above. In addition to serine-threonine kinases, thedescribed NHPs also share significant similarity to several additionalkinase families, including kinases associated with signal transduction,from a variety of phyla and species. Several polymorphisms wereidentified in the described NHPs. These include a T/C polymorphism inthe sequence region represented by nucleotide position 1170 of SEQ IDNO:1, both of which result in the same amino acid being present at thecorresponding amino acid (aa) position of SEQ ID NO:2; a T/Cpolymorphism in the sequence region represented by nucleotide position1321 of SEQ ID NO:1, both of which result in the same amino acid beingpresent at the corresponding aa position of SEQ ID NO:2; a C/Gpolymorphism in the sequence region represented by nucleotide position94 of SEQ ID NO:3, which can result in either a leu or val being presentat corresponding aa position 32 of SEQ ID NO:4; an A/G polymorphism atnucleotide position 112 of SEQ ID NO:3, which can result in either a lysor glu being present at corresponding aa position 38 of SEQ ID NO:4; andan A/T polymorphism at nucleotide position 133 of SEQ ID NO:3, which canresult in either a thr or ser being present at corresponding aa position45 of SEQ ID NO:4. The above polymorphisms can be present either singly,or in any combination or permutation within a given sequence.

[0049] An additional application of the described novel humanpolynucleotide sequences is their use in the molecularmutagenesis/evolution of proteins that are at least partially encoded bythe described novel sequences using, for example, polynucleotideshuffling or related methodologies. Such approaches are described inU.S. Pat. Nos. 5,830,721 and 5,837,458, which are herein incorporated byreference in their entirety.

[0050] NHP gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, worms, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, birds, goats, andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate NHP transgenic animals.

[0051] Any technique known in the art may be used to introduce a NHPtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to, pronuclearmicroinjection (Hoppe and Wagner, 1989, U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); gene targeting inembryonic stem cells (Thompson et al., 1989, Cell 56:313-321);electroporation of embryos (Lo, 1983, Mol Cell. Biol. 3:1803-1814); andsperm-mediated gene transfer (Lavitrano et al., 1989, Cell 57:717-723);etc. For a review of such techniques, see Gordon, 1989, TransgenicAnimals, Intl. Rev. Cytol. 115:171-229, which is incorporated byreference herein in its entirety.

[0052] The present invention provides for transgenic animals that carrythe NHP transgene in all their cells, as well as animals that carry thetransgene in some, but not all their cells, i.e., mosaic animals orsomatic cell transgenic animals. The transgene may be integrated as asingle transgene or in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al., 1992, Proc. Natl. Acad. Sci. USA89:6232-6236. The regulatory sequences required for such a cell-typespecific activation will depend upon the particular cell type ofinterest, and will be apparent to those of skill in the art.

[0053] When it is desired that a NHP transgene be integrated into thechromosomal site of the endogenous NHP gene, gene targeting ispreferred. Briefly, when such a technique is to be utilized, vectorscontaining some nucleotide sequences homologous to the endogenous NHPgene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous NHP gene (i.e.,“knockout” animals).

[0054] The transgene can also be selectively introduced into aparticular cell type, thus inactivating the endogenous NHP gene in onlythat cell type, by following, for example, the teaching of Gu et al.,1994, Science, 265:103-106. The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

[0055] Once transgenic animals have been generated, the expression ofthe recombinant NHP gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques that include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of NHP gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the NHPtransgene product.

5.2 NHPS AND NHP POLYPEPTIDES

[0056] NHPs, NHP polypeptides, NHP peptide fragments, mutated,truncated, or deleted forms of the NHPs, and/or NHP fusion proteins canbe prepared for a variety of uses. These uses include, but are notlimited to, the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products relatedto a NHP, as reagents in assays for screening for compounds that can beused as pharmaceutical reagents useful in the therapeutic treatment ofmental, biological, or medical disorders and disease. Given thesimilarity information and expression data, the described NHPs can betargeted (by drugs, oligos, antibodies, etc.) in order to treat disease,or to therapeutically augment the efficacy of therapeutic agents.

[0057] The Sequence Listing discloses the amino acid sequences encodedby the described NHP-encoding polynucleotides. The NHPs displayinitiator methionines that are present in DNA sequence contextsconsistent with eucaryotic translation initiation sites. The NHPs do notdisplay consensus signal sequences, which indicates that they may becytoplasmic or possibly nuclear proteins, however, the homology data andpresence of hydrophobic domains indicates that the NHPs are probablymembrane associated, or possibly secreted.

[0058] The NHP amino acid sequences of the invention include the aminoacid sequences presented in the Sequence Listing as well as analoguesand derivatives thereof. Further, corresponding NHP homologues fromother species are encompassed by the invention. In fact, any NHP proteinencoded by the NHP nucleotide sequences described above are within thescope of the invention, as are any novel polynucleotide sequencesencoding all or any novel portion of an amino acid sequence presented inthe Sequence Listing. The degenerate nature of the genetic code iswell-known, and, accordingly, each amino acid presented in the SequenceListing, is generically representative of the well-known nucleic acid“triplet” codon, or in many cases codons, that can encode the aminoacid. As such, as contemplated herein, the amino acid sequencespresented in the Sequence Listing, when taken together with the geneticcode (see, for example, Table 4-1 at page 109 of “Molecular CellBiology”, 1986, J. Darnell et al., eds., Scientific American Books, NewYork, N.Y., herein incorporated by reference) are genericallyrepresentative of all the various permutations and combinations ofnucleic acid sequences that can encode such amino acid sequences.

[0059] The invention also encompasses proteins that are functionallyequivalent to the NHPs encoded by the presently described nucleotidesequences, as judged by any of a number of criteria, including, but notlimited to, the ability to bind and modify a NHP substrate, or theability to effect an identical or complementary downstream pathway, or achange in cellular metabolism (e.g., proteolytic activity, ion flux,tyrosine phosphorylation, etc.). Such functionally equivalent NHPproteins include, but are not limited to, additions or substitutions ofamino acid residues within the amino acid sequence encoded by the NHPnucleotide sequences described above, but that result in a silentchange, thus producing a functionally equivalent expression product.Amino acid substitutions may be made on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. For example, nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid.

[0060] A variety of host-expression vector systems can be used toexpress the NHP nucleotide sequences of the invention. Where the NHPpeptide or polypeptide can exist, or has been engineered to exist, as asoluble or secreted molecule, the soluble NHP peptide or polypeptide canbe recovered from the culture media. Such expression systems alsoencompass engineered host cells that express a NHP, or functionalequivalent, in situ. Purification or enrichment of a NHP from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well-known to those skilled in the art.However, such engineered host cells themselves may be used in situationswhere it is important not only to retain the structural and functionalcharacteristics of the NHP, but to assess biological activity, e.g., indrug screening assays.

[0061] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining NHP nucleotide sequences; yeast (e.g., Saccharomyces, Pichia)transformed with recombinant yeast expression vectors containing NHPnucleotide sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing NHP nucleotidesequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing NHP nucleotide sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing NHP nucleotide sequences and promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter).

[0062] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the NHPproduct being expressed. For example, when a large quantity of such aprotein is to be produced for the generation of pharmaceuticalcompositions of or containing NHP, or for raising antibodies to a NHP,vectors that direct the expression of high levels of fusion proteinproducts that are readily purified may be desirable. Such vectorsinclude, but are not limited to, the E. coli expression vector pUR278(Ruther et al., 1983, EMBO J. 2:1791), in which a NHP coding sequencemay be ligated individually into the vector in frame with the lacZcoding region so that a fusion protein is produced; pIN vectors (Inouyeand Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke andSchuster, 1989, J. Biol. Chem. 264:5503-5509); and the like. pGEXvectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetexpression product can be released from the GST moiety.

[0063] In an insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) can be used as a vector to express foreign polynucleotidesequences. The virus grows in Spodoptera frugiperda cells. A NHP codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of NHP coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted sequence is expressed (e.g., see Smith etal., 1983, J. Virol. 46:584; Smith, U.S. Pat. No. 4,215,051).

[0064] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the NHP nucleotide sequence of interest may beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericsequence may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing a NHP product in infected hosts(e.g., see Logan and Shenk, 1984, Proc. Natl. Acad. Sci. USA81:3655-3659). Specific initiation signals may also be required forefficient translation of inserted NHP nucleotide sequences. Thesesignals include the ATG initiation codon and adjacent sequences. Incases where an entire NHP gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of a NHP coding sequenceis inserted, exogenous translational control signals, including,perhaps, the ATG initiation codon, may be provided. Furthermore, theinitiation codon should be in phase with the reading frame of thedesired coding sequence to ensure translation of the entire insert.These exogenous translational control signals and initiation codons canbe of a variety of origins, both natural and synthetic. The efficiencyof expression may be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (seeBitter et al.; 1987, Methods in Enzymol. 153:516-544).

[0065] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes theexpression product in the specific fashion desired. Such modifications(e.g., glycosylation) and processing (e.g., cleavage) of proteinproducts may be important for the function of the protein. Differenthost cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins andexpression products. Appropriate cell lines or host systems can bechosen to ensure the desired modification and processing of the foreignprotein expressed. To this end, eukaryotic host cells that possess thecellular machinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the expression product may beused. Such mammalian host cells include, but are not limited to, CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, humancell lines.

[0066] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the NHP sequences described above can be engineered. Rather thanusing expression vectors that contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci, which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express the NHPproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the NHP product.

[0067] A number of selection systems may be used, including, but notlimited to, the herpes simplex virus thymidine kinase (Wigler et al.,1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase(Szybalska and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), andadenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817)genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for the following genes: dhfr, which confers resistance tomethotrexate (Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77:3567;O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, whichconfers resistance to mycophenolic acid (Mulligan and Berg, 1981, Proc.Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.150:1); and hygro, which confers resistance to hygromycin (Santerre etal., 1984, Gene 30:147).

[0068] Alternatively, any fusion protein can be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, an exemplary system allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the sequence of interest is subcloned into a vaccinia recombinationplasmid such that the sequence's open reading frame is translationallyfused to an amino-terminal tag consisting of six histidine residues.Extracts from cells infected with recombinant vaccinia virus are loadedonto Ni²+-nitriloacetic acid-agarose columns and histidine-taggedproteins are selectively eluted with imidazole-containing buffers.

[0069] Also encompassed by the present invention are fusion proteinsthat direct the NHP to a target organ and/or facilitate transport acrossthe membrane into the cytosol. Conjugation of NHPs to antibody moleculesor their Fab fragments could be used to target cells bearing aparticular epitope. Attaching the appropriate signal sequence to the NHPwould also transport the NHP to the desired location within the cell.Alternatively, targeting of NHP or its nucleic acid sequence might beachieved using liposome or lipid complex based delivery systems. Suchtechnologies are described in “Liposomes: A Practical Approach”, New,R.R.C., ed., Oxford University Press, New York, and in U.S. Pat. Nos.4,594,595, 5,459,127, 5,948,767 and 6,110,490 and their respectivedisclosures, which are herein incorporated by reference in theirentirety. Additionally embodied are novel protein constructs engineeredin such a way that they facilitate transport of the NHP to the targetsite or desired organ, where they cross the cell membrane and/or thenucleus where the NHP can exert its functional activity. This goal maybe achieved by coupling of the NHP to a cytokine or other ligand thatprovides targeting specificity, and/or to a protein transducing domain(see generally U.S. Provisional Patent Application Ser. Nos. 60/111,701and 60/056,713, both of which are herein incorporated by reference, forexamples of such transducing sequences) to facilitate passage acrosscellular membranes, and can optionally be engineered to include nuclearlocalization.

5.3 ANTIBODIES TO NHP PRODUCTS

[0070] Antibodies that specifically recognize one or more epitopes of aNHP, or epitopes of conserved variants of a NHP, or peptide fragments ofa NHP, are also encompassed by the invention. Such antibodies include,but are not limited to, polyclonal antibodies, monoclonal antibodies(mAbs), humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0071] The antibodies of the invention can be used, for example, in thedetection of NHP in a biological sample and may, therefore, be utilizedas part of a diagnostic or prognostic technique whereby patients may betested for abnormal amounts of NHP. Such antibodies may also be utilizedin conjunction with, for example, compound screening schemes for theevaluation of the effect of test compounds on expression and/or activityof a NHP expression product. Additionally, such antibodies can be usedin conjunction gene therapy to, for example, evaluate the normal and/orengineered NHP-expressing cells prior to their introduction into thepatient. Such antibodies may additionally be used as a method for theinhibition of abnormal NHP activity. Thus, such antibodies may,therefore, be utilized as part of treatment methods.

[0072] For the production of antibodies, various host animals may beimmunized by injection with the NHP, a NHP peptide (e.g., onecorresponding to a functional domain of a NHP), truncated NHPpolypeptides (NHP in which one or more domains have been deleted),functional equivalents of the NHP, or mutated variants of the NHP. Suchhost animals may include, but are not limited to, pigs, rabbits, mice,goats, and rats, to name but a few. Various adjuvants may be used toincrease the immunological response, depending on the host species,including, but not limited to, Freund's adjuvant (complete andincomplete), mineral salts such as aluminum hydroxide or aluminumphosphate, chitosan, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Alternatively, the immune response could beenhanced by combination and or coupling with molecules such as keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, ovalbumin, choleratoxin or fragments thereof. Polyclonal antibodies are heterogeneouspopulations of antibody molecules derived from the sera of the immunizedanimals.

[0073] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique (Kohler and Milstein, 1975, Nature 256:495-497; andU.S. Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosboret al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl.Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole etal., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96). Such antibodies may be of any immunoglobulin class,including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Thehybridoma producing the mAbs of this invention may be cultivated invitro or in vivo. Production of high titers of mAbs in vivo makes thisthe presently preferred method of production.

[0074] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA,81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608-; Takeda etal., 1985, Nature, 314:452-454) by splicing the genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion. Such technologies are described in U.S. Pat. Nos. 6,075,181 and5,877,397 and their respective disclosures, which are hereinincorporated by reference in their entirety. Also encompassed by thepresent invention is the use of fully humanized monoclonal antibodies,as described in U.S. Pat. No. 6,150,584 and respective disclosures,which are herein incorporated by reference in their entirety.

[0075] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA85:5879-5883; and Ward et al., 1989, Nature 341:544-546) can be adaptedto produce single chain antibodies against NHP expression products.Single chain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide.

[0076] Antibody fragments that recognize specific epitopes may begenerated by known techniques. For example, such fragments include, butare not limited to: F(ab′)₂ fragments, which can be produced by pepsindigestion of the antibody molecule; and Fab fragments, which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0077] Antibodies to a NHP can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” a given NHP, using techniqueswell-known to those skilled in the art (see, e.g., Greenspan and Bona,1993, FASEB J. 7:437-444; and Nissinoff, 1991, J. Immunol.147:2429-2438). For example, antibodies that bind to a NHP domain andcompetitively inhibit the binding of NHP to its cognate receptor/ligandcan be used to generate anti-idiotypes that “mimic” the NHP and,therefore, bind, activate, or neutralize a NHP, NHP receptor, or NHPligand. Such anti-idiotypic antibodies, or Fab fragments of suchanti-idiotypes, can be used in therapeutic regimens involving a NHPmediated pathway.

[0078] Additionally given the high degree of relatedness of mammalianNHPs, the presently described knock-out mice (having never seen a NHP,and thus never been tolerized to a NHP) have a unique utility, as theycan be advantageously applied to the generation of antibodies againstthe disclosed mammalian NHPs (i.e., a NHP will be immunogenic in NHPknock-out animals).

[0079] The present invention is not to be limited in scope by thespecific embodiments described herein, which are intended as singleillustrations of individual aspects of the invention, and functionallyequivalent methods and components are within the scope of the invention.Indeed, various modifications of the invention, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the foregoing description. Such modifications are intended tofall within the scope of the appended claims. All cited publications,patents, and patent applications are herein incorporated by reference intheir entirety.

1 5 1 2079 DNA homo sapiens 1 atggagaagt acgagcggat ccgagtggtggggagaggtg ccttcgggat tgtgcacctg 60 tgcctgcgaa aggctgacca gaagctggtgatcatcaagc agattccagt ggaacagatg 120 accaaggaag agcggcaggc agcccagaatgagtgccagg tcctcaagct gctcaaccac 180 cccaatgtca ttgagtacta cgagaacttcctggaagaca aagcccttat gatcgccatg 240 gaatatgcac caggcggcac tctggctgagttcatccaaa agcgctgtaa ttccctgctg 300 gaggaggaga ccatcctgca cttcttcgtgcagatcctgc ttgcactgca tcatgtgcac 360 acccacctca tcctgcaccg agacctcaagacccagaaca tcctgcttga caaacaccgc 420 atggtcgtca agatcggtga tttcggcatctccaagatcc ttagcagcaa gagcaaggcc 480 tacacggtgg tgggtacccc atgctatatctcccctgagc tgtgtgaggg caagccctac 540 aaccagaaga gtgacatctg ggccctgggctgtgtcctct acgagctggc cagcctcaag 600 agggctttcg aggctgcgaa cttgccagcactggtgctga agatcatgag tggcaccttt 660 gcacctatct ctgaccggta cagccctgagcttcgccagc tggtcctgag tctactcagc 720 ctggagcctg cccagcggcc accactcagccacatcatgg cacagcccct ctgcatccgt 780 gccctcctca acctccacac cgacgtgggcagtgtccgca tgcggagggc agagaagtcc 840 gtggccccca gcaacacagg gagcaggaccaccagtgtcc gctgcagagg tatcccccgg 900 ggacctgtga ggccagccat cccaccaccactgtcgtcag tgtatgcctg gggtggtggg 960 ctgggcaccc ccctgcggct gccaatgctcaacacagagg tggtccaggt ggcagctggg 1020 cgcacgcaga aagccggcgt cacgcgctctgggcgtctca tcctgtggga ggccccaccc 1080 ctaggtgcag gcggaggcag tctccttcctggggcagtgg agcagccaca gccccagttc 1140 atctcgcgtt tcctggaggg ccagtcgggygtgaccatca agcacgtggc ctgtggggac 1200 ttcttcactg cctgcctgac tgacagaggcatcatcatga cattcggcag cggcagcaat 1260 gggtgcctag gccatggcag cctcactgacatcagccagc ccaccattgt ggaggctttg 1320 ytgggctatg aaatggtgca ggtggcctgtggggcctctc acgtgctggc cctgtccact 1380 gagcgagaac tatttgcctg gggccgtggagacagcggca gactggggct aggcaccagg 1440 gagtcccaca gctgccccca gcaggtgcccatgcccccag gacaggaagc tcagcgagtt 1500 gtatgtggta tcgattcctc catgatcctcactgtgcctg gccaagccct agcctgtggg 1560 agcaacaggt tcaacaagct gggcctggaccacctctccc tgggggagga gcctgtcccc 1620 caccagcaag tggaggaggc cctgagcttcacactactag gctctgcacc cctggaccag 1680 gagcctctgc tgagtataga cctgggcactgctcactcag ctgctgtgac tgcctcgggt 1740 gattgctaca cttttggcag caatcagcacggacagttgg gcaccaatac tcgccgaggc 1800 agtcgggcac cctgtaaggt ccaaggccttgagggcatca agatggcaat ggtagcctgt 1860 ggggatgcct tcactgtagc tattggggcagagagcgaag tgtactcttg gggcaaaggg 1920 gcgcgaggtc gattgggaag gagggatgaggatgccggac tccctcggcc agtgcagttg 1980 gatgagacac acccttacac ggtgacttccgtgtcctgtt gccatggaaa caccctcctg 2040 gctgttcgat cggtcacaga tgagccggtccccccctga 2079 2 692 PRT homo sapiens 2 Met Glu Lys Tyr Glu Arg Ile ArgVal Val Gly Arg Gly Ala Phe Gly 1 5 10 15 Ile Val His Leu Cys Leu ArgLys Ala Asp Gln Lys Leu Val Ile Ile 20 25 30 Lys Gln Ile Pro Val Glu GlnMet Thr Lys Glu Glu Arg Gln Ala Ala 35 40 45 Gln Asn Glu Cys Gln Val LeuLys Leu Leu Asn His Pro Asn Val Ile 50 55 60 Glu Tyr Tyr Glu Asn Phe LeuGlu Asp Lys Ala Leu Met Ile Ala Met 65 70 75 80 Glu Tyr Ala Pro Gly GlyThr Leu Ala Glu Phe Ile Gln Lys Arg Cys 85 90 95 Asn Ser Leu Leu Glu GluGlu Thr Ile Leu His Phe Phe Val Gln Ile 100 105 110 Leu Leu Ala Leu HisHis Val His Thr His Leu Ile Leu His Arg Asp 115 120 125 Leu Lys Thr GlnAsn Ile Leu Leu Asp Lys His Arg Met Val Val Lys 130 135 140 Ile Gly AspPhe Gly Ile Ser Lys Ile Leu Ser Ser Lys Ser Lys Ala 145 150 155 160 TyrThr Val Val Gly Thr Pro Cys Tyr Ile Ser Pro Glu Leu Cys Glu 165 170 175Gly Lys Pro Tyr Asn Gln Lys Ser Asp Ile Trp Ala Leu Gly Cys Val 180 185190 Leu Tyr Glu Leu Ala Ser Leu Lys Arg Ala Phe Glu Ala Ala Asn Leu 195200 205 Pro Ala Leu Val Leu Lys Ile Met Ser Gly Thr Phe Ala Pro Ile Ser210 215 220 Asp Arg Tyr Ser Pro Glu Leu Arg Gln Leu Val Leu Ser Leu LeuSer 225 230 235 240 Leu Glu Pro Ala Gln Arg Pro Pro Leu Ser His Ile MetAla Gln Pro 245 250 255 Leu Cys Ile Arg Ala Leu Leu Asn Leu His Thr AspVal Gly Ser Val 260 265 270 Arg Met Arg Arg Ala Glu Lys Ser Val Ala ProSer Asn Thr Gly Ser 275 280 285 Arg Thr Thr Ser Val Arg Cys Arg Gly IlePro Arg Gly Pro Val Arg 290 295 300 Pro Ala Ile Pro Pro Pro Leu Ser SerVal Tyr Ala Trp Gly Gly Gly 305 310 315 320 Leu Gly Thr Pro Leu Arg LeuPro Met Leu Asn Thr Glu Val Val Gln 325 330 335 Val Ala Ala Gly Arg ThrGln Lys Ala Gly Val Thr Arg Ser Gly Arg 340 345 350 Leu Ile Leu Trp GluAla Pro Pro Leu Gly Ala Gly Gly Gly Ser Leu 355 360 365 Leu Pro Gly AlaVal Glu Gln Pro Gln Pro Gln Phe Ile Ser Arg Phe 370 375 380 Leu Glu GlyGln Ser Gly Val Thr Ile Lys His Val Ala Cys Gly Asp 385 390 395 400 PhePhe Thr Ala Cys Leu Thr Asp Arg Gly Ile Ile Met Thr Phe Gly 405 410 415Ser Gly Ser Asn Gly Cys Leu Gly His Gly Ser Leu Thr Asp Ile Ser 420 425430 Gln Pro Thr Ile Val Glu Ala Leu Leu Gly Tyr Glu Met Val Gln Val 435440 445 Ala Cys Gly Ala Ser His Val Leu Ala Leu Ser Thr Glu Arg Glu Leu450 455 460 Phe Ala Trp Gly Arg Gly Asp Ser Gly Arg Leu Gly Leu Gly ThrArg 465 470 475 480 Glu Ser His Ser Cys Pro Gln Gln Val Pro Met Pro ProGly Gln Glu 485 490 495 Ala Gln Arg Val Val Cys Gly Ile Asp Ser Ser MetIle Leu Thr Val 500 505 510 Pro Gly Gln Ala Leu Ala Cys Gly Ser Asn ArgPhe Asn Lys Leu Gly 515 520 525 Leu Asp His Leu Ser Leu Gly Glu Glu ProVal Pro His Gln Gln Val 530 535 540 Glu Glu Ala Leu Ser Phe Thr Leu LeuGly Ser Ala Pro Leu Asp Gln 545 550 555 560 Glu Pro Leu Leu Ser Ile AspLeu Gly Thr Ala His Ser Ala Ala Val 565 570 575 Thr Ala Ser Gly Asp CysTyr Thr Phe Gly Ser Asn Gln His Gly Gln 580 585 590 Leu Gly Thr Asn ThrArg Arg Gly Ser Arg Ala Pro Cys Lys Val Gln 595 600 605 Gly Leu Glu GlyIle Lys Met Ala Met Val Ala Cys Gly Asp Ala Phe 610 615 620 Thr Val AlaIle Gly Ala Glu Ser Glu Val Tyr Ser Trp Gly Lys Gly 625 630 635 640 AlaArg Gly Arg Leu Gly Arg Arg Asp Glu Asp Ala Gly Leu Pro Arg 645 650 655Pro Val Gln Leu Asp Glu Thr His Pro Tyr Thr Val Thr Ser Val Ser 660 665670 Cys Cys His Gly Asn Thr Leu Leu Ala Val Arg Ser Val Thr Asp Glu 675680 685 Pro Val Pro Pro 690 3 2454 DNA homo sapiens 3 atgcccgccgccactccagc cccgcagccg ccgccgcccc cggcccggcc agccccagcc 60 tgcccggcgcggcctgcccc gggacagcaa ggcctatgtg accattctct aaaatattta 120 agctcgagaatcacagagcg gaagctgcaa ggctcctggc tgcctgccag ccgagggaat 180 ctggagaaaccattcctggg gccgcgtggc cccgtcgtgc ccttgttctg ccctcggaat 240 ggccttcactcagcacatcc tgagaacagc cctctgaagc ccagggtcgt gaccgtagtg 300 aagctgggtgggcagcgccc ccgaaagatc actctgctcc tcaacaggcg atcagtgcag 360 acgttcgagcagctcttagc tgacatctca gaagccttgg gctctcccag atggaagaat 420 gaccgtgtgaggaaactgtt taacctcaag ggcagggaaa tcaggagcgt ctctgatttc 480 ttcagggaaggggatgcttt catagctatg ggcaaagaac cactgacact gaagagcatt 540 caggtggctgtagaagaact gtaccccaac aaagcccggg ccctgacact ggcccagcac 600 agccgtgccccttctccaag gctgaggagc aggctgttta gcaaggctct gaaaggagac 660 caccgctgtggggagaccga gacccccaag agctgcagcg aagttgcagg atgcaaggca 720 gccatgaggcaccaggggaa gatccccgag gagctttcac tagatgacag agcgaggacc 780 cagaagaagtgggggagggg gaaatgggag ccagaaccca gtagcaagcc ccccagggaa 840 gccactctggaagagaggca cgcaagggga gagaagcatc ttggggtgga gattgaaaag 900 acctcgggtgaaattatcag atgcgagaag tgcaagagag agagggagct tcagcagagc 960 ctggagcgtgagaggctttc tctggggacc agtgagctgg atatggggaa gggcccaatg 1020 tatgatgtggagaagctggt gaggaccaga agctgcagga ggtctcccga ggcaaatcct 1080 gcaagtggggaggaagggtg gaagggtgac agccacagga gcagccccag gaatcccact 1140 caagagctgaggagacccag caagagcatg gacaagaaag aggacagagg cccagaggat 1200 caagaaagccatgctcaggg agcagccaag gccaagaagg accttgtgga agttcttcct 1260 gtcacagaggaggggctgag ggaggtgaag aaggacacca ggcccatgag caggagcaaa 1320 catggtggctggctcctgag agagcaccag gcgggctttg agaagctccg caggacccga 1380 ggagaagagaaggaggcaga gaaggagaaa aagccatgta tgtctggagg cagaaggatg 1440 actctcagagatgaccaacc tgcaaagcta gaaaaggagc ccaagacgag gccagaagag 1500 aacaagccagagcggcccag cggtcggaag ccacggccca tgggcatcat tgccgccaat 1560 gtggaaaagcattatgagac tggccgggtc attggggatg ggaactttgc tgtcgtgaag 1620 gagtgcagacaccgcgagac caggcaggcc tatgcgatga agatcattga caagtccaga 1680 ctcaagggcaaggaggacat ggtggacagt gagatcttga tcatccagag cctctctcac 1740 cccaacatcgtgaaattgca tgaagtctac gaaacagaca tggaaatcta cctgatcctg 1800 gagtacgtgcagggaggaga cctttttgac gccatcatag aaagtgtgaa gttcccggag 1860 cccgatgctgccctcatgat catggactta tgcaaagccc tcgtccacat gcacgacaag 1920 agcattgtccaccgggacct caagccggaa aaccttttgg ttcagcgaaa tgaggacaaa 1980 tctactaccttgaaattggc tgattttgga cttgcaaagc atgtggtgag acctatattt 2040 actgtgtgtgggaccccaac ttacgtagct cccgaaattc tttctgagaa aggttatgga 2100 ctggaggtggacatgtgggc tgctggcgtg atcctctata tcctgctgtg tggctttccc 2160 ccattccgcagccctgagag ggaccaggac gagctcttta acatcatcca gctgggccac 2220 tttgagttcctcccccctta ctgggacaat atctctgatg ctgctaaaga tctggtgagc 2280 cggttgctggtggtagaccc caaaaagcgc tacacagctc atcaggttct tcagcacccc 2340 tggatcgaaacagctggcaa gaccaataca gtgaaacgac agaagcaggt gtcccccagc 2400 agcgatggtcacttccggag ccagcacaag agggttgtgg agcaggtatc atag 2454 4 817 PRT homosapiens 4 Met Pro Ala Ala Thr Pro Ala Pro Gln Pro Pro Pro Pro Pro AlaArg 1 5 10 15 Pro Ala Pro Ala Cys Pro Ala Arg Pro Ala Pro Gly Gln GlnGly Leu 20 25 30 Cys Asp His Ser Leu Lys Tyr Leu Ser Ser Arg Ile Thr GluArg Lys 35 40 45 Leu Gln Gly Ser Trp Leu Pro Ala Ser Arg Gly Asn Leu GluLys Pro 50 55 60 Phe Leu Gly Pro Arg Gly Pro Val Val Pro Leu Phe Cys ProArg Asn 65 70 75 80 Gly Leu His Ser Ala His Pro Glu Asn Ser Pro Leu LysPro Arg Val 85 90 95 Val Thr Val Val Lys Leu Gly Gly Gln Arg Pro Arg LysIle Thr Leu 100 105 110 Leu Leu Asn Arg Arg Ser Val Gln Thr Phe Glu GlnLeu Leu Ala Asp 115 120 125 Ile Ser Glu Ala Leu Gly Ser Pro Arg Trp LysAsn Asp Arg Val Arg 130 135 140 Lys Leu Phe Asn Leu Lys Gly Arg Glu IleArg Ser Val Ser Asp Phe 145 150 155 160 Phe Arg Glu Gly Asp Ala Phe IleAla Met Gly Lys Glu Pro Leu Thr 165 170 175 Leu Lys Ser Ile Gln Val AlaVal Glu Glu Leu Tyr Pro Asn Lys Ala 180 185 190 Arg Ala Leu Thr Leu AlaGln His Ser Arg Ala Pro Ser Pro Arg Leu 195 200 205 Arg Ser Arg Leu PheSer Lys Ala Leu Lys Gly Asp His Arg Cys Gly 210 215 220 Glu Thr Glu ThrPro Lys Ser Cys Ser Glu Val Ala Gly Cys Lys Ala 225 230 235 240 Ala MetArg His Gln Gly Lys Ile Pro Glu Glu Leu Ser Leu Asp Asp 245 250 255 ArgAla Arg Thr Gln Lys Lys Trp Gly Arg Gly Lys Trp Glu Pro Glu 260 265 270Pro Ser Ser Lys Pro Pro Arg Glu Ala Thr Leu Glu Glu Arg His Ala 275 280285 Arg Gly Glu Lys His Leu Gly Val Glu Ile Glu Lys Thr Ser Gly Glu 290295 300 Ile Ile Arg Cys Glu Lys Cys Lys Arg Glu Arg Glu Leu Gln Gln Ser305 310 315 320 Leu Glu Arg Glu Arg Leu Ser Leu Gly Thr Ser Glu Leu AspMet Gly 325 330 335 Lys Gly Pro Met Tyr Asp Val Glu Lys Leu Val Arg ThrArg Ser Cys 340 345 350 Arg Arg Ser Pro Glu Ala Asn Pro Ala Ser Gly GluGlu Gly Trp Lys 355 360 365 Gly Asp Ser His Arg Ser Ser Pro Arg Asn ProThr Gln Glu Leu Arg 370 375 380 Arg Pro Ser Lys Ser Met Asp Lys Lys GluAsp Arg Gly Pro Glu Asp 385 390 395 400 Gln Glu Ser His Ala Gln Gly AlaAla Lys Ala Lys Lys Asp Leu Val 405 410 415 Glu Val Leu Pro Val Thr GluGlu Gly Leu Arg Glu Val Lys Lys Asp 420 425 430 Thr Arg Pro Met Ser ArgSer Lys His Gly Gly Trp Leu Leu Arg Glu 435 440 445 His Gln Ala Gly PheGlu Lys Leu Arg Arg Thr Arg Gly Glu Glu Lys 450 455 460 Glu Ala Glu LysGlu Lys Lys Pro Cys Met Ser Gly Gly Arg Arg Met 465 470 475 480 Thr LeuArg Asp Asp Gln Pro Ala Lys Leu Glu Lys Glu Pro Lys Thr 485 490 495 ArgPro Glu Glu Asn Lys Pro Glu Arg Pro Ser Gly Arg Lys Pro Arg 500 505 510Pro Met Gly Ile Ile Ala Ala Asn Val Glu Lys His Tyr Glu Thr Gly 515 520525 Arg Val Ile Gly Asp Gly Asn Phe Ala Val Val Lys Glu Cys Arg His 530535 540 Arg Glu Thr Arg Gln Ala Tyr Ala Met Lys Ile Ile Asp Lys Ser Arg545 550 555 560 Leu Lys Gly Lys Glu Asp Met Val Asp Ser Glu Ile Leu IleIle Gln 565 570 575 Ser Leu Ser His Pro Asn Ile Val Lys Leu His Glu ValTyr Glu Thr 580 585 590 Asp Met Glu Ile Tyr Leu Ile Leu Glu Tyr Val GlnGly Gly Asp Leu 595 600 605 Phe Asp Ala Ile Ile Glu Ser Val Lys Phe ProGlu Pro Asp Ala Ala 610 615 620 Leu Met Ile Met Asp Leu Cys Lys Ala LeuVal His Met His Asp Lys 625 630 635 640 Ser Ile Val His Arg Asp Leu LysPro Glu Asn Leu Leu Val Gln Arg 645 650 655 Asn Glu Asp Lys Ser Thr ThrLeu Lys Leu Ala Asp Phe Gly Leu Ala 660 665 670 Lys His Val Val Arg ProIle Phe Thr Val Cys Gly Thr Pro Thr Tyr 675 680 685 Val Ala Pro Glu IleLeu Ser Glu Lys Gly Tyr Gly Leu Glu Val Asp 690 695 700 Met Trp Ala AlaGly Val Ile Leu Tyr Ile Leu Leu Cys Gly Phe Pro 705 710 715 720 Pro PheArg Ser Pro Glu Arg Asp Gln Asp Glu Leu Phe Asn Ile Ile 725 730 735 GlnLeu Gly His Phe Glu Phe Leu Pro Pro Tyr Trp Asp Asn Ile Ser 740 745 750Asp Ala Ala Lys Asp Leu Val Ser Arg Leu Leu Val Val Asp Pro Lys 755 760765 Lys Arg Tyr Thr Ala His Gln Val Leu Gln His Pro Trp Ile Glu Thr 770775 780 Ala Gly Lys Thr Asn Thr Val Lys Arg Gln Lys Gln Val Ser Pro Ser785 790 795 800 Ser Asp Gly His Phe Arg Ser Gln His Lys Arg Val Val GluGln Val 805 810 815 Ser 5 2824 DNA homo sapiens 5 cgggctcgtg gctgctcgtctcgccccgcc ttcccgcgcc tgctcgaccg tcgagccgcg 60 tccccgcgct gccacctctgctccaggctc tccccgagcc cgccgccgcg ccatgcccgc 120 cgccactcca gccccgcagccgccgccgcc cccggcccgg ccagccccag cctgcccggc 180 gcggcctgcc ccgggacagcaaggcctatg tgaccattct ctaaaatatt taagctcgag 240 aatcacagag cggaagctgcaaggctcctg gctgcctgcc agccgaggga atctggagaa 300 accattcctg gggccgcgtggccccgtcgt gcccttgttc tgccctcgga atggccttca 360 ctcagcacat cctgagaacagccctctgaa gcccagggtc gtgaccgtag tgaagctggg 420 tgggcagcgc ccccgaaagatcactctgct cctcaacagg cgatcagtgc agacgttcga 480 gcagctctta gctgacatctcagaagcctt gggctctccc agatggaaga atgaccgtgt 540 gaggaaactg tttaacctcaagggcaggga aatcaggagc gtctctgatt tcttcaggga 600 aggggatgct ttcatagctatgggcaaaga accactgaca ctgaagagca ttcaggtggc 660 tgtagaagaa ctgtaccccaacaaagcccg ggccctgaca ctggcccagc acagccgtgc 720 cccttctcca aggctgaggagcaggctgtt tagcaaggct ctgaaaggag accaccgctg 780 tggggagacc gagacccccaagagctgcag cgaagttgca ggatgcaagg cagccatgag 840 gcaccagggg aagatccccgaggagctttc actagatgac agagcgagga cccagaagaa 900 gtgggggagg gggaaatgggagccagaacc cagtagcaag ccccccaggg aagccactct 960 ggaagagagg cacgcaaggggagagaagca tcttggggtg gagattgaaa agacctcggg 1020 tgaaattatc agatgcgagaagtgcaagag agagagggag cttcagcaga gcctggagcg 1080 tgagaggctt tctctggggaccagtgagct ggatatgggg aagggcccaa tgtatgatgt 1140 ggagaagctg gtgaggaccagaagctgcag gaggtctccc gaggcaaatc ctgcaagtgg 1200 ggaggaaggg tggaagggtgacagccacag gagcagcccc aggaatccca ctcaagagct 1260 gaggagaccc agcaagagcatggacaagaa agaggacaga ggcccagagg atcaagaaag 1320 ccatgctcag ggagcagccaaggccaagaa ggaccttgtg gaagttcttc ctgtcacaga 1380 ggaggggctg agggaggtgaagaaggacac caggcccatg agcaggagca aacatggtgg 1440 ctggctcctg agagagcaccaggcgggctt tgagaagctc cgcaggaccc gaggagaaga 1500 gaaggaggca gagaaggagaaaaagccatg tatgtctgga ggcagaagga tgactctcag 1560 agatgaccaa cctgcaaagctagaaaagga gcccaagacg aggccagaag agaacaagcc 1620 agagcggccc agcggtcggaagccacggcc catgggcatc attgccgcca atgtggaaaa 1680 gcattatgag actggccgggtcattgggga tgggaacttt gctgtcgtga aggagtgcag 1740 acaccgcgag accaggcaggcctatgcgat gaagatcatt gacaagtcca gactcaaggg 1800 caaggaggac atggtggacagtgagatctt gatcatccag agcctctctc accccaacat 1860 cgtgaaattg catgaagtctacgaaacaga catggaaatc tacctgatcc tggagtacgt 1920 gcagggagga gacctttttgacgccatcat agaaagtgtg aagttcccgg agcccgatgc 1980 tgccctcatg atcatggacttatgcaaagc cctcgtccac atgcacgaca agagcattgt 2040 ccaccgggac ctcaagccggaaaacctttt ggttcagcga aatgaggaca aatctactac 2100 cttgaaattg gctgattttggacttgcaaa gcatgtggtg agacctatat ttactgtgtg 2160 tgggacccca acttacgtagctcccgaaat tctttctgag aaaggttatg gactggaggt 2220 ggacatgtgg gctgctggcgtgatcctcta tatcctgctg tgtggctttc ccccattccg 2280 cagccctgag agggaccaggacgagctctt taacatcatc cagctgggcc actttgagtt 2340 cctcccccct tactgggacaatatctctga tgctgctaaa gatctggtga gccggttgct 2400 ggtggtagac cccaaaaagcgctacacagc tcatcaggtt cttcagcacc cctggatcga 2460 aacagctggc aagaccaatacagtgaaacg acagaagcag gtgtccccca gcagcgatgg 2520 tcacttccgg agccagcacaagagggttgt ggagcaggta tcatagtcac caccttggga 2580 atctgtccag cccccagttctgctcaagga cagagaaaag gatagaagtt tgagagaaaa 2640 acaatgaaag aggcttcttcacataattgg tgaatcagag ggagagacac tgagtatatt 2700 ttaaagcata ttaaaaaaattaagtcaatg ttaaatgtca caacatattt ttagatttgt 2760 atatttaaag cctttaatacatttttgggg ggtaagcatt gtcatcagtg aggaattttg 2820 gtaa 2824

What is claimed is:
 1. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1.
 2. An isolated nucleic acid moleculecomprising a nucleotide sequence that encodes the amino acid sequenceshown in SEQ ID NO:2.
 3. An isolated nucleic acid molecule comprising atleast 24 contiguous bases from SEQ ID NO:3.
 4. An isolated nucleic acidmolecule comprising a nucleotide sequence that: (a) encodes the aminoacid sequence shown in SEQ ID NO:3; and (b) hybridizes under highlystringent conditions to the nucleotide sequence of SEQ ID NO:4 or thecomplement thereof.
 5. An isolated nucleic acid expression vectorcomprising a promoter element operatively positioned to express atranscript encoding the amino acid sequence shown in SEQ ID NO:4.